European Journal of Medicinal Chemistry 108 (2016) 79e88

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Anti-herpetic and anti-dengue activity of abietane ferruginol

analogues synthesized from (þ)-dehydroabietylamine
Vicky C. Roa-Linares a, b, Yaneth M. Brand a, b, Lee S. Agudelo-Gomez a,

nica Tangarife-Castan
Vero ~ o a, Liliana A. Betancur-Galvis a, b, Juan C. Gallego-Gomez b,

lez c , *
Miguel A. Gonza
a
Group of Investigative Dermatology, Institute of Medical Research, Medicine Faculty, University of Antioquia, Medellin, A.A1226, Antioquia, Colombia
b
Translational and Molecular Medicine Group, Institute of Medical Research, Medicine Faculty, University of Antioquia, Medellin, Colombia
c
Departamento de Química Orga
nica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The abietane-type diterpenoid (þ)-ferruginol (1), a bioactive compound isolated from several plants, has
Received 11 August 2015 attracted much attention as consequence of its pharmacological properties, which includes antibacterial,
Received in revised form antifungal, antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-
23 September 2015
ulcerogenic, anti-inflammatory and antitumor actions. In this study, we report on the antiviral evalua-
Accepted 5 November 2015
Available online 11 November 2015
tion of ferruginol (1) and several analogues synthesized from commercial (þ)-dehydroabietylamine.
Thus, the activity against Human Herpesvirus type 1, Human Herpesvirus type 2 and Dengue Virus type
2, was studied. Two ferruginol analogues showed high antiviral selectivity index and reduced viral
Keywords:
Antiviral
plaque-size in post-infection stages against both Herpes and Dengue viruses. A promising lead, com-
Herpes pound 8, was ten-fold more potent (EC50 ¼ 1.4 mM) than the control ribavirin against Dengue Virus type
Dengue 2. Our findings suggest that the 12-hydroxyabieta-8,11,13-triene skeleton, which is characteristic of the
Abietane diterpenoid ferruginol (1), is an interesting molecular scaffold for development of novel antivirals. In
Diterpene addition, the cytotoxic and antifungal activities of the synthesized ferruginol analogues have also been
Ferruginol investigated. ©20155 Elsevier Science. All rights reserved.
Dehydroabietylamine © 2015 Elsevier Masson SAS. All rights reserved.

1. Introduction
Human Herpesvirus (HHV) types 1 and 2 are enveloped DNA
viruses characterized for producing latent infection in sensory neu-
rons and reactivation during periods of severe immunosuppression
of the host. Many factors such as stress, fatigue, sexual relations with
people who have active lesions, excess exposure to heat or cold, fe-
ver, laser treatments, local tissue trauma and nerve damage, can
predispose to reactivation, especially in neonates, transplant and
immunocompromised patients which tend to present aggressive and
recurrent lesions [1]. Also, it has been reported that prolonged
therapy with Acyclovir (ACV), and its analogues, has induced the
emergence of drug-resistant virus strains. Human Herpesvirus pre-
dominantly develops resistance, almost 95%, as a result of mutations
in the genes that encode the viral thymidine kinase (TK) [2].
* Corresponding author.
E-mail address: Miguel.A.Gonzalez@uv.es (M.A. Gonz

alez).
However, mutations in the viral DNA polymerase can also cause
this resistance. In immunocompromised patients, the presence of
HHV species resistant to ACV, with a prevalence of 4e10%, has
complicated their clinical management [3]. Moreover, Dengue virus
(DENV) is an enveloped ssRNA virus. To date, there have been
described four serotypes established in humans (DENV-1 to DENV-
4) and recently, a fifth Dengue subtype that follows the sylvatic
cycle has been identified [4]. Dengue is the most important
mosquito-borne disease worldwide, since it is distributed over one
hundred countries of the tropical belt and the tropics, both the Old
and New World [5]. Disease manifestations cover a wide spectrum
ranging from dengue with or without warning signs to severe
dengue in which plasma leakage, haemorrhage and organ impair-
ment can lead to death. In addition, unusual manifestations such as
cardiomyopathy, liver failure and neurological disorders have been
reported [6]. The major problem of this pathology is that there are
no drugs approved for human treatment or vaccine available for
prophylaxis. Also, antiviral drugs addressed to specific genes or
proteins of the virus are not a good alternative due to the high

http://dx.doi.org/10.1016/j.ejmech.2015.11.009
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
80 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
mutation rate of RNA viruses, often because viral mutants are
selected for drug resistance [7]. During the last years in Colombia,
several reports had shown the high rates of evolution of DENV,
possibly related with the new Asiatic-American genotypes found in
Colombia and Brazil [8].
The aromatic abietane-type diterpenoids comprise a large
number of natural metabolites of plant origin which possess a wide
variety of biological effects [9]. These abietanes have been the
target of several synthetic campaigns towards both the natural
products and synthetic derivatives with interesting pharmacolog-
ical properties [10]. Among these known bioactive compounds,
(þ)-ferruginol (1), some derivatives of (þ)-dehydroabietic acid (2)
and (þ)-dehydroabietylamine (3) as well as (þ)-jiadifenoic acid C
(4) (Fig. 1) have shown promising results, including antiviral
properties [10b,11].
The present study is a continuation of our research programs to
discover bioactive diterpenoids [12]. It includes the synthesis of a
focused library based on (þ)-ferruginol (1) from commercially
available (þ)-dehydroabietylamine (3), where a series of phthali-
mides could have potential antiviral activity [13]. A fullerene
diterpenoid hybrid was also envisaged as potential inhibitor [14].
Herein, we describe the antiviral activity of ferruginol (1) and some
analogues (5e14) (Scheme 1) synthesized from dehydroabietyl-
amine (3) against HHV-1 and HHV-2, and DENV-2. Our aim is to
identify new antiviral drug candidates which act on cellular and/or
molecular targets instead on viral proteins. In this way, the con-
cerns about the viral mutants resistant to the treatments can be
avoided. This approach has been recently called host-targeted
antiviral in an evolutionary study of resistance of Dengue viruses
under selective pressure of several antiviral agents [15].
2. Results and discussion
2.1. Chemistry
The synthesis of (þ)-ferruginol (1) and analogues 5e14 from
commercial (þ)-dehydroabietylamine (3) was performed as out-
lined in the Scheme 1. Compounds 1 and 5e9 were synthesized as
reported in the literature using a FriedeleCrafts acylation and a
BaeyereVilliger oxidation as key steps [16]. Then, compound 6 was
used as starting material for the preparation of compounds 10 and
11. Later, compound 11 was converted into compounds 12e14 as
described recently in the literature [17]. Thus, the synthesis starts
with the introduction of the phthalimide group on (þ)-dehy-
droabietylamine (3), followed by FriedeleCrafts acylation and
oxidation under Baeyer-Villiger conditions to afford acetate 5 in
72% overall yield. Hydrolysis of the acetate group in 5 gave phenol 8
in high yield, while overall deprotection of 5 afforded the amino-
phenol 6 in 75% yield. Compound 6 was the intermediate of three
separate approaches. Firstly, tosylation under standard conditions
gave compound 7 in 90% yield. Secondly, oxidative deamination of
Fig. 1. Examples of bioactive aromatic abietane diterpenoids.
6 gave 18-oxoferruginol (9) in moderate yield (50%), which was
converted into ferruginol (1) by Wolff-Kishner reduction (90%
yield). And finally, the treatment of 6 with tetrachlorophthalic an-
hydride (TCPA) afforded phenol 10 in 75% yield, which was acety-
lated with acetic anhydride in pyridine to give acetate 11 in
quantitative yield. Acetate 11 was oxidized at C-7 with excess of t-
BuOOH as oxidant and CrO3/pyridine mixture as a catalyst in DCM,
the yield of ketone 12 was 66%. Subsequently, the reaction of 12
with p-tosylhydrazide yielded the corresponding p-tosylhydrazone
13 (77% yield). The fullerene-terpenoid hybrid 14 was obtained by
the treatment with NaOMe of 13 in anhydrous pyridine for 20 min
at room temperature followed by addition of a solution of C60 in
chlorobenzene and heating at 70  C for 24 h. This gave compound
14 in ca. 30% yield. All the compounds showed spectroscopic data in
agreement with the assigned structures and purity >95%.
2.2. Biological evaluation
The synthesized compounds 1 and 5e14 (Scheme 1) were
evaluated for antiviral activity against HHV-1, HHV-2 and DENV-2
(see Table 1). Two ferruginol analogues, compounds 8 and 9 (18-
oxoferruginol), showed relevant activity.
In particular, the ferruginol analogue 8 showed moderate ac-
tivity against HHV-1 (Rf ¼ 1  102) at a concentration of 14.5 mM,
high activity against HHV-2 (Rf ¼ 1  103) and reduction of cyto-
pathic effect during DENV-2 infection, comparable to control of
untreated cells, at concentration of 29.0 mM. Compound 9 (18-
oxoferruginol) also showed high activity against HHV-2
(Rf ¼ 1  103) at a concentration of 41.7 mM, however, its ability
to exert reduction of cytopathic effect in the presence of DENV-2
infection was lower than that of compound 8. Moreover, com-
pound 9 did not show activity against HHV-1.
In 2007, Wen and co-workers reported the antiviral potential of
certain diterpenoid derivatives, including the abietane-type diter-
penoid ferruginol (1), during in vitro coronavirus infection [18].
However, at present, the antiviral activity of the abietane class of
compounds has not been much investigated, especially the study of
derivatives and analogues; therefore, the activity against HHV-1,
HHV-2 and DENV-2 reported in this study is to our knowledge,
the first report of antiviral activity of ferruginol analogues. Also, as
the compounds 8 and 9 are active against two different viruses,
such as herpesvirus and dengue virus, our findings suggest that
these molecules probably have a broad-spectrum of antiviral ac-
tivity against enveloped viruses with DNA and RNA genome.
Later on, to check the specific antiviral activity of these com-
pounds, we carried out the evaluation of other biological activities
such as cytotoxic and antifungal, because is well known that fer-
ruginol (1) has promising bioactivities, such as antifungal, anti-
bacterial, miticidal antiplasmodial, antileishmanial, nematicidal,
and antitumor, among others [9]. In Table 2, it is shown the cyto-
toxic activity on Vero cells as well as on the tumor cells HeLa, Jurkat
and U937 of ferruginol 1 and analogues 5e14.
Compounds 1, 6, 7, 9, 10 and 12 produced a dose-dependent
inhibition on the growth of the three tumor cell lines: Jurkat,
U937 and HeLa, as well as the Vero cell line, with R2 (coefficient of
linear regression) > 0.8. Most of these compounds showed cyto-
toxic activity against at least one tumor cell line at concentrations
below 30 mM. However, only compound 10 against HeLa and Jurkat
cell lines, and compound 12 against the three tumor cell lines,
showed a great SI (SI  5), being compound 12 the most selective
agent against tumor cell lines respect the non-tumor cell line
(SI > 43.8, >26.0 and > 38.5, for HeLa, Jurkat and U937, respec-
tively). The compounds 5, 8, 11, 13 and 14 were not active against
the tumor cell lines at the tested concentrations and hence they
were not evaluated on the Vero cell line.
 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88 81

Scheme 1. Synthesis of the tested compounds 1 and 5e14.

In addition, the antifungal activity of all compounds was studied Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus. Only
against two dermatophytes (Trichophyton rubrum and Trichophyton one ferruginol analogue, compound 9, showed anti-dermatophyte
mentagrophytes), and the filamentous fungi Fusarium oxysporum, activity against T. rubrum and T. mentagrophytes with geometric

Table 1
Reduction of viral titer, inhibition of cytopathic effect and antiviral activity against HHV-1, HHV-2 and DENV-2 of ferruginol analogues 8 and 9.

Vero cell linea

Compound HHV-1 HHV-2 DENV-2

Rfb Antiviral activity (mM)c Rfb Antiviral activity (mM)c CPE inhibitiond Antiviral activity (mM)c

8 102 14.5 103 29.0 þ 29.0

9 NA NA 103 41.7 þ 41.7
ACV 104 6.7 103 6.7 NT NT
RIBA NT NT NT NT þ 123.0

HHV-1: Human Herpesvirus type 1 (CDC Atlanta strain). HHV-2: Human Herpesvirus type 2 (VR-734-G strain). DENV-2: Dengue virus (New Guinea strain). NT: not tested. NA:
not active.
These values represent the mean of two independent experiments.
a
Vero Cercopithecus aethiops African green monkey kidney cell line ATCC CCL-81.
b
Reduction factor of the viral titer.
c
Maximal non-toxic concentration that showed viral reduction factor.
d
Cytopathic effect inhibition (þ).
82 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88

Table 2
Cytotoxicity (IC50-mM) of ferruginol (1) and analogues 5e14 on HeLa, Jurkat, U937 and Vero cell lines.

Compound Cell linesc

HeLa Jurkat U937 Vero

IC50a (mM) SI b
IC50a (mM) SI b
IC50a (mM) SI b
IC50a (mM)

1 64.9 1.4 48.2 1.9 21.3 4.2 90.4

5 >50 NA >50 NA >50 NA NE
6 >80 NA 49.4 0.8 >80 NA 39.8
7 >50 NA 30.9 2.7 36.0 2.3 82.5
8 >50 NA >50 NA >50 NA NE
9 60.0 1.5 21.6 4.2 33.2 2.7 90.8
10 29.0 9.2 21.9 12.2 >40 NA 266.8
11 >40 NA >40 NA >40 NA NE
12 7.3 >43.8 12.3 >26.0 8.3 >38.5 >320
13 >30 NA >30 NA >30 NA NE
14 >30 NA >30 NA >30 NA NE
PXT 0.099 NA 0.0005 NA 0.006 NA 0.65
DOX 0.834 NA 0.012 NA 0.077 NA 1.93

PXT: Paclitaxel; DOX: Doxorubicine. NA: Not apply; NE: Not evaluated. These values represent the geometric mean of two independent experiments, each one made by
quadruplicate.
a
Inhibitory concentration 50 defined as concentration of compounds that induces 50% of growth inhibition at 48 h.
b
SI, selectivity index defined as VERO IC50 over either Jurkat, U937 or HeLa IC50.
c
Jurkat, human acute T cell leukemia ATCC TIB-152; U937, human promonocytic cell line ATCC CRL-1593.2; HeLa, human cervix epitheloid adenocarcinoma cells ATCC CRL-
1958; Vero, Cercopithecus aethiops African green monkey kidney cells ATCC CCL-81.

mean of minimal inhibitory concentration (GM-MIC) values of
41.7 mM and 83.3 mM, respectively. None of the compounds showed
activity against the filamentous fungus tested (data not shown).
Likewise, none of the compounds showed anti-Candida activity
against three yeast strains (Candida albicans, Candida parapsilosis
and Candida tropicalis) (data not shown).
Although there are several studies showing that ferruginol (1)
and some related abietanes exhibit promising antifungal and
cytotoxic activity [19], our results reveals that compound 8 lacks of
antifungal activity in the fungal models tested and cytotoxicity
against HeLa, Jurkat and U937 tumor cells at the evaluated con-
centrations. Therefore, this compound could be a selective mole-
cule against enveloped viruses.
2.3. Mechanism of antiviral activity of ferruginol analogues 8 and 9
Previous studies suggest that the mechanism by which ferru-
ginol (1) exerts antitumor activity is related to the enhanced
expression of BAX proapoptotic protein, inhibition of signaling
pathways involved in cell survival and proliferation (Ras /PI3K and
Jak/STAT) and reduction of expression of cyclin-dependent kinases
[20]. However, the cellular and molecular basis by which these
compounds exhibit antiviral activity has not been currently
described.
In order to define the stage of viral replication cycle in which the
ferruginol analogues 8 and 9 were active, we performed two
different conditions of treatment. Firstly, we tested the effect of
compounds on early stages of viral replication cycle, such as cell-
free viral particle, adhesion and viral entry by treatment before
infection (pre-infection treatment). And secondly, we tested the
antiviral activity on several steps of viral cycle that includes traffic,
replication, assembly, egress of new viral particles and cell-to-cell
spread by treatment after infection (post-infection treatment).
As can be seen in Table 3, compounds 8 and 9 were effective
during post-infection treatment and were not active during pre-
infection treatment.
With both pre- and post-infection treatments, a plaque reduc-
tion assay was performed and the concentration of compounds 8
and 9 that reduced the number of viral plaques in 50% (EC50) was
interpolated from the doseeresponse curves. Furthermore, the
antiviral selectivity index (SI) was calculated through the relation
between the inhibitory concentration 50 (IC50) in Vero cells and
EC50 of each virus (IC50/EC50) (Table 3).
Compound 8 reduced the 50% of plaque forming units at a
concentration of 19.2 mM against HHV-2 and showed a high SI equal
to 10, whereas compound 9 reduced the number of plaques in 50%
at concentrations of 19.6 mM and 16.6 mM, for HHV-1 and HHV-2,
respectively. As expected the controls, DEX-S and ACV, did show
antiviral activity in pre-treatment and post-treatment, respectively.
These results show that the compounds 8 and 9 have important
activity against HHV-2 strain at low concentrations, in comparison
to HHV-1. This is possibly due to a smaller number of receptors
available in the host cell to carry out the infection with HHV-2,
making this virus more sensitive to the action of the compounds
[21].
Differences in the activity of these compounds were found
against HHV-1 strain. In the end-point titration technique (EPTT)
assay, compound 8 showed moderate activity (Table 1, Rf ¼ 1  102)
and in the plaque forming unit (PFU) assay this compound did not
show a dose-dependent effect. However, it showed a significant
reduction in viral plaque size (Table 3, highlighted with an asterisk),
related among others, with a low rate of viral replication and
reduction of cytopathic effect, confirming the antiviral effect of this
compound.
On the other hand, compound 9 did not show reduction of viral
titer during screening (EPTT) against HHV-1, but inhibits the
number of plaque forming units during the PFU assay. This differ-
ence can be explained due the amount of virus used in each assay.
In the EPTT assay a high amount of virus (10 TCID50) was used and
the concentration of compound that showed antiviral activity could
not be detected. In the PFU assay, 100 PFU/well was used and the
EC50 value was determined, nevertheless, this compound did not
show a high SI value (4.9), demonstrating closeness between the
cytotoxic concentration and the antiviral concentration.
Likewise, the anti-dengue activity of these ferruginol analogues
was evaluated 8 days post-infection (d.p.i), being both compounds
8 and 9 active at EC50 concentrations of 1.4 mM (SI ¼ 57.7) and
5.0 mM (SI ¼ 10.4), respectively.
The antiviral selectivity index is defined as the relative effec-
tiveness of a product to inhibit viral replication compared to the
capacity for inducing cell death. This index results useful to make
bioactivity comparisons between the compounds and helps in
 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
Table 3
Antiviral activity (EC50-mM) on pre and post-infection treatment of ferruginol analogues 8 and 9.
Compound IC50 IC50 EC50 (mM) (SI)
(mM) (mM) Pre-infection treatment
72 h 8 days
HHV-1 HHV-2
8 190.7 80.8 NI NI
9 97.8 52.2 NI NI
DEX-S >10 NT 0.015 0.012
ACV >1700 NT NT NT
RIBA NT NT NT NT
EC50, 50% antiviral effective concentration; IC50, 50% inhibitory concentration of cell proliferation (mM); SI, Antiviral selectivity index values (IC50/EC50) are presented between
parenthesis; DEX-S, dextran sulfate; ACV, acyclovir; RIBA, ribavirin; *Reduction on viral plaque-size. NI, no inhibitory activity; NT, not tested. These values represent the mean
of three independent experiments.
designing more potent compounds. In recent reports, SI values
higher than 10 (SI > 10) are considered indicative of a potential
therapeutic agent that would merit further biopharmaceutical and
pre-clinical studies [22]. Therefore, compound 8 is promissory to
continue this type of study.
Additionally to the dose-dependent effect previously described
during post-infection treatment, we found that compounds 8 and 9
induced a reduction on viral plaque size. To determine the average
of reduction of viral plaque size during compound-treatment
relative to mock-treated control, we performed an image analysis
using Image-Pro Plus 6.0 software (Fig. 2). The area of plaques was
measured in millimeters and statistically significant differences (P
value < 0.001) were found in all cases. Compound 8 showed a
dramatic reduction on viral plaque size (93.7%) at a concentration
of 29.0 mM against HHV-1 (CDC-Atlanta acyclovir-sensitive strain)
(Fig. 2A). Respect to HHV-2, compound 9 reduced the size of viral
plaques (68.2%) at a concentration of 20.8 mM (Fig. 2B).
Furthermore, during treatment with compound 8 at a concen-
tration of 29.0 mM (Fig. 2C) on cell infection by HHV-1 acyclovir-
resistant strain 29R (thymidine kinase mutant), we found a similar
effect to that showed against the acyclovir-sensitive strain,
reducing the viral plaque size in 87.1%. This finding may suggest
that the phosphorylation by viral thymidine kinase it is not
required for the effect exerted by this compound, thus, is possible
that anti-herpetic mechanism of action of ferruginol analogue 8
differs to acyclovir and other nucleoside analogues.
Likewise, reduction on viral plaque-size (77.9%) was observed
when cells infected with DENV-2 were treated with compound 8 at
concentration of 3.6 mM (Fig. 2D). The reduction percentages re-
ported for all viruses indicate the reduction of viral plaque size
during compound-treatment relative to mock-treated control
(100%). Representations of plaque morphology of HHV-1(CDC
Atlanta), HHV-1 (29R) and DENV-2 after the treatment with com-
pound 8 and mock-treated control, are shown in Figures 2E, F and
2H, respectively. Fig. 2G shows the effect after treatment with
compound 9 against HHV-2 and mock-treated control.
The reduction on viral plaque size of HHV strains and DENV-2 in
the presence of compounds 8 and 9 suggests that these ferruginol
analogues may prevent the first-round of viral replication, release
and/or cell-to-cell spread, being this last stage an important factor
in infectivity, virulence, establishment of latency and immune
response evasion during Herpesvirus infections [23]. Therefore,
inhibition of viral intercellular diffusion is an attractive target in the
search for new antiviral drugs, mainly to treat the latent infections
and drug-resistant strains.
In recent years, several compounds have shown effects on virus
cell-to-cell spreading during Herpesvirus infection. For example,
Lin and co-workers reported that chebulagic acid and punicalagin,
two hydrolyzable tannins isolated from of Terminalia chebula
(Combretaceae), inhibit the HHV-1 cell-to-cell spread. This fact was
83
EC50 (mM) (SI)
Post-infection treatment
DENV-2 HHV-1 HHV-2 DENV-2
NI NI* 19.2 (10.0) 1.4 (57.7)*
NI 19.6 (4.9) 16.6 (5.9)* 5.0 (10.4)
NT NT NT NT
NT 2.2 0.27 NT
NT NT NT 13.5
verified 24 h post-treatment using a fluorescent plaque assay in the
presence of HHV-1 neutralizing antibodies to guarantee cell-to-cell
transmission via intercellular junctions between infected and un-
infected cells [24]. Meanwhile, Nyberg and Ekblad and co-workers
demonstrated that the low molecular weight heparin sulfate-
mimic, PI-88, which is a mixture of highly sulfated mannose con-
taining oligosaccharides, reduced the area of viral plaques with IC50
values of 2 mg/mL and 0.7 mg/mL for HHV-1 and HHV-2, respectively
[25]. Nevertheless, to date it has not been reported a similar effect
on viral plaque size of HHV and other viruses from abietane diter-
penoids, among these, ferruginol (1) and their derivatives.
Several molecular and cellular components involved in
Herpesvirus cell-to-cell spread have been previously reported. For
example, viral glycoproteins such as E and M play a relevant role in
normal viral plaque phenotype of HHV-1, because the plaque size of
the double-deletion mutant (DgEgM) reduced over 5-fold less than
wild type strain [26]. In polarized epithelial cells, Johnson and co-
workers demonstrated by electron microscopy, that the gE/gI gly-
coproteins complex is necessary to accumulate virions at cell
junctions, since mutants that not express gE glycoprotein leads to
particles accumulation more frequently on apical surfaces. Thus,
they concluded that the gE/gI complex is required to sort selectively
HHV-1 particles to lateral surfaces and specifically to cell junctions
[27]. On the other hand, structures containing actin and tubulin
during Herpesviruses infection have the ability to project virions
towards adjacent cells. Additionally, both the membrane compo-
sition and cytoskeletal structures of egress sites are modified dur-
ing infection in non-polarized cells [28]. According to the findings
of Dixit and co-workers, HHV-1 also promotes cytoskeletal rear-
rangements that facilitate viral spread in vitro, including dramatic
increases of filopodia in cultured neurons [29]. Multiple and
convergent ways by which several viruses such as Vaccinia, Hepa-
titis B and C viruses, Human Immunodeficiency virus, Human T-
lymphotropic virus, Murine leukemia virus and HHV cause sub-
version of cytoskeleton for different objectives in viral cycle are
summarized by Taylor and co-workers [30]. The actin cytoskeleton
is reorganized by these viruses affecting every stage of the viral life
cycle, from entry through assembly to egress. Using immuno-
chemical assays, drug inhibition assays and protein interaction
profiling methods, Wang and co-workers identified the ways in
which DENV-2 interacts with actin cytoskeleton. This study showed
that dynamic treadmilling of actin is necessary for Dengue virus
entry, production and release, indicating a direct effect of viral E
protein on the structural modifications of actin cytoskeleton [31].
Considering that cytoskeletal components such as microtubules
and actin microfilaments are required for both Herpesvirus and
Flavivirus replication cycle (mainly involved in the entry, traffic,
release and cell-to-cell spread), is possible that ferruginol ana-
logues 8 and 9 cause disruption of these cellular pathways which
may be involved in viral spread. Nonetheless, other experiments
Fig. 2. Effect of compounds 8 and 9 on HHV-1, HHV-2 and DENV-2 plaque-size during Vero cell infection. Effect on viral plaque-size of compounds 8 and 9 in presence of 100 PFU/
mL of HHV-1 (2A), HHV-2 (2B), HHV-1 29R strain (2C) and DENV-2 (2D). Results were expressed as an average of plaque size area (mm) of 80 viral plaques developed in compound-
treated cells relative to mock-treated controls. Statistically significant differences were found in all cases with p values < 0.001 (***). Parallel, the image analysis using Image-Pro
Plus 6.0 software represents the reduction of viral plaque size during treatment with compounds 8 and 9 of HHV-1 (2E), HHV-2 (2F), HHV-1 29R strain (2G) and DENV-2 (2H). Two
separate experiments were carried out for each compound.
 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
are required to confirm this hypothesis.
Although it is very risky assure that some viral or cellular
component are involved in our findings, the study of ferruginol
analogues will be an important tool from medicinal chemistry for
dissecting these complicated cellular and molecular processes of
the viral infections. In the future, this research will be routed to
understanding of some cellular and molecular mechanisms where,
two very different viruses (Herpesvirus and Dengue), are
converging in a conserved evolutionary pathway during cell
infection, affecting cytoskeleton elements, endomembrane system
and signaling pathways involved in viral egress from the host-cells
and spreading infection toward neighbor cells.
3. Conclusions
In summary, we have discovered important antiviral properties
in the molecular scaffold 12-hydroxyabieta-8,11,13-triene func-
tionalized at C-18, which is reminiscent of the abietane diterpenoid
ferruginol (1). Two ferruginol analogues, compounds 8 and 9,
synthesized from readily available (þ)-dehydroabietylamine (3)
were found to consistently inhibit Human Herpesvirus (type 1 and
2) and Dengue virus type 2 at low micromolar concentrations.
These compounds showed antiviral activity specifically in post-
infective stages and significantly reduced the viral plaque-size
during infection. The presence of a phthalimide moiety in the
molecule, compound 8, resulted in the highest antiviral selectivity
index against the viruses tested and a ten-fold more potency
(EC50 ¼ 1.4 mM) than the control ribavirin against Dengue Virus
type 2.
With regard to the cytotoxic properties of the tested com-
pounds, it should be noted that the chlorinated-phthalimide
analogue 12 exhibited moderate cytotoxicity of broad spectrum,
without toxicity towards the non-tumor cell line (Vero). Addi-
tionally, the tested compounds did not show relevant antifungal
activity.
Based on the above, the results here reported are relevant for
further research of possible cellular and molecular mechanisms
involved in the effect of reduction on viral plaque size. Also, these
findings are important to promote the realization of biopharma-
ceutical testing and finding new highly selective antiviral drugs
mainly directed towards cellular targets.
4. Experimental
4.1. Chemistry
4.1.1. General procedures
Optical rotations were measured using a 5 cm cell in a Schmidt-
Haensch Polartronic-D polarimeter. NMR spectra were recorded on
a 300 MHz spectrometer. All spectra were recorded in CDCl3 as
solvent unless otherwise stated. Complete assignments of 13C NMR
multiplicities were made on the basis of DEPT experiments. J values
are given in Hz. MS data were acquired on a QTOF spectrometer.
Reactions were monitored by TLC using Merck silica gel 60 F-254 in
0.25 mm-thick plates. Compounds on TLC plates were detected
under UV light at 254 nm and visualized by immersion in a 10%
sulfuric acid solution and heating with a heat gun. Purifications
were performed by flash chromatography on Merck silica gel
(230e400 mesh). Commercial reagent grade solvents and chem-
icals were used as purchased unless otherwise noted. Combined
organic extracts were washed with brine, dried over anhydrous
NaSO4, filtered, and concentrated under reduced pressure.
4.1.2. Materials
Compounds 1 and 5e9 have been prepared according to known
85
procedures [16]. Compound 6 was used as starting material for the
preparation of compounds 10 and 11. Later, compound 11 was
converted into compounds 12e14 as described recently in the
literature [17]. All compounds prepared in this work exhibit spec-
troscopic data in agreement with the proposed structures. Purity of
all final compounds was 95% or higher.
4.1.2.1. 12-Hydroxy-N,N- (tetrachlorophthaloyl)
dehydroabietylamine (10)
A mixture of the tetrachlorophthalic anhydride (640 mg,
2.14 mmol) and 12-hydroxydehydroabietylamine 6 [16a] (647 mg,
2.14 mmol) in acetic acid (7.5 mL) was heated at reflux for 2 h, then
cooled to rt and 50 mL of water was added. The resulting solid was
filtered off, washed with water, dried under vacuum and purified by
column chromatography eluting with toluene to give compound 10
(913 mg, 75%) as a yellow solid: [a]20 1
D 10.0 (c 1.0, CHCl3); H NMR
(300 MHz) d 6.87 (1H, s), 6.60 (1H, s), 3.67 (1H, d, J ¼ 15.0), 3.54 (1H,
d, J ¼ 15.0), 3.10 (1H, m), 2.92 (2H, m), 2.15 (2H, m), 1.24 (3H, d,
J ¼ 6.0), 1.22 (3H, d, J ¼ 6.0), 1.22 (3H, s), 1.04 (3H, s); 13C NMR
(75 MHz) dC 2  164.5 (s), 150.6 (s), 148.1 (s), 2  140.1 (s), 131.6 (s),
2  129.5 (s), 2  127.4 (s), 127.1 (s), 126.8 (d), 110.5 (d), 49.8 (t), 45.2
(d), 39.5 (s), 38.0 (t), 37.5 (s), 37.1 (t), 29.3 (t), 26.7 (d), 25.7 (q), 22.7
(q), 22.5 (q), 19.5 (t), 19.0 (q), 18.4 (t). HRMS (ESI) m/z 568.0986
[Mþ1]þ, calcd for C28H30Cl4NO3: 568.0980.
4.1.2.2. 12-Acetoxy-N,N-(tetrachlorophthaloyl)
dehydroabietylamine (11)
A solution of phenol 10 (656 mg, 1.15 mmol) in a 1:1 mixture
(14 mL) of acetic anhydride and pyridine was stirred at rt for one
day. Then, 150 mL of water were added and the mixture was
extracted with ethyl acetate (3  25 mL). The combined organic
extracts were washed with 6% NaHCO3 (2  50 mL), water and
brine. The resulting organic extract was dried over sodium sulfate,
filtered and concentrated. The residue was chromatographed on
silica eluting with chloroform to afford acetate 11 (704 mg, 100%) as
a white solid with identical spectroscopic data to the reported in
the literature [17].
4.3. Biology
4.3.1. Reagents and compounds
Dulbecco's Modified Eagle's Medium (DMEM) and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
were obtained from SigmaeAldrich Chemical Co. (St. Louis, MO,
USA). Fetal bovine serum (FBS) and penicillin/streptomycin were
purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).
Acyclovir was obtained from Biogen Laboratory. Ribavirin was ob-
tained from Calbiochem (La Jolla, CA, USA). Paclitaxel, Doxor-
ubicine, Amphotericine B and Itraconazole were purchased from
SigmaeAldrich Chemical Co. Terbinafine was obtained from
Recalcine Laboratories (Santiago de Chile, Chile). Dimethyl sulf-
oxide (DMSO) was purchased from Merck KGaA (Darmstadt,
Germany).
Stock solutions of compounds were prepared in DMSO and
frozen at 70  C. The concentration of DMSO in biological assays
was of 0.05%. Cell controls with DMSO at 0.05% were used.
4.3.2. Cell culture and viruses
Human Herpesvirus type 1 (HHV-1 CDC Atlanta acyclovir-
sensitive strain) was obtained from the Center for Disease Control
(Atlanta, GA, USA); HHV-1 29R (acyclovir-resistant strain) was
donated by Prof. Claudia Oliveira (University of Santacatarina-
Brazil) and Human Herpesvirus type 2 (HHV-2 VR-734-G
acyclovir-sensitive strain) was obtained from the Center for Dis-
ease Control. All of them were amplified in Vero cells (African green
86 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
monkey kidney-Cercopithecus aethiops, ATCC: CCL 81 line). Virus
stocks were titrated in Vero cells by plaque assay and expressed as
plaque forming units (PFU/mL). Dengue virus type 2 (DENV-2 New
Guinea strain) was donated by Maria Elena Pen ~ aranda and Eva
Harris (Sustainable Sciences Institute and the University of Cali-
fornia at Berkeley) was amplified in C6/36HT of Aedes albopictus
cells, from ATCC, and titrated in Vero cells following our laboratory
conditions [32].
Cells were maintained in Dulbecco's Modified Eagle Medium
(DMEM) supplemented with 5% of inactivated FBS to Vero cells and
10% of inactivated FBS to C6/36HT cells, 100 units/mL of penicillin,
100 mg/mL of streptomycin, 100 mg/mL of L-glutamine, 0.14%
NaHCO3, and 1% of each non-essential amino acids and minimum
essential medium vitamin solution (choline chloride, D-calcium
pantothenate, folic acid, nicotinamide, pyridoxal hydrochloride,
riboflavin, thiamine hydrochloride and i-inositol). Vero cells were
incubated at 37  C in humidified 5% CO2 atmosphere and C6/36 HT
at 34  C in humidified 5% CO2 atmosphere.
To determine the cytotoxic activity, cell lines of human cervix
epitheloid adenocarcinoma cells (HeLa, ATCC CCL-2), acute T cell
leukemia (Jurkat, ATCC TIB-152) and human promonocytic cell line
(U937, ATCC CRL-1593.2) were used, as well as, the non-tumor cell
line Vero. Vero and HeLa cells were grown in DMEM supplemented.
Jurkat and U937 cells were maintained in RPMI-1640 medium
(supplemented with 10% FBS), 100 units/mL of penicillin, 100 mg/mL
of streptomycin, and 100 mg/mL of neomycin and maintained at
37  C in humidified 5% CO2 atmosphere.
4.4. Antiviral activity
4.4.1. Screening against HHV-1 and HHV-2
The anti-herpetic activity of ferruginol analogues on Vero cells
against 10 TCID50 of HHV-1 and HHV-2, were carried out using the
end-point titration technique (EPTT). Vero cells were harvested in
96-well plates at a density of 2.0  104 cells/well at 37  C in hu-
midified 5% CO2 atmosphere until constituted 80% of the cell
monolayer. Viral suspension/compound mixture were performed
in DMEM with 2% FBS supplemented containing carboxymethyl-
cellulose (CMC) to 1% and 0.5% for HHV-1 and HHV-2, respectively
and incubated for 15 min at room temperature. Compounds were
evaluated at concentrations of 6.25e50 mg/mL. After 48 h (HHV-1)
and 72 h (HHV-2) of incubation at 37  C in a humidified 5% CO2
atmosphere, the cell monolayers were stained with a solution of
3.5% formaldehyde with 0.2% crystal violet and the plaques were
counted. Two independent experiments by quadruplicate were
carried out for each viral serotype and each compound. Controls
were included untreated cells, cells treated with compounds and
cells infected with HHV-1 or HHV-2. Positive control included in
this assay was Acyclovir. According to the parameters established
by Vlietinck et al. [33] the relevant or moderate antiviral activity of
a purified natural product, is one whose reduction factor (Rf) of viral
titer is respectively of  1  103 or 1  102. This parameter was used
in our study.
4.4.2. Screening against DENV-2
To determine the anti-DENV activity we carried out the protocol
previously described by Tang et al. [34] with some modifications.
Briefly, Vero cells were harvested in 96-well plates at a density of
2.0  104 cells/well at 37  C in humidified 5% CO2 atmosphere. After
24 h, medium was removed and two-fold serial dilutions of DENV-2
at 10TCID50 and non-cytotoxic concentrations of compounds were
incubated for 10 min at room temperature. Then, 50 mL of each
dilution (compound/DENV-2) was added and the plates were
incubated at 37  C for 3 days, time at which the cytopathic effect
(CPE) in control (infected cells) was observed in 100% of cell
monolayer. The cell monolayers were stained with a solution of
3.5% formaldehyde with 0.2% crystal violet and CPE was observed
under inverted microscope. Controls were included untreated cells,
cells treated with compounds and cells infected with DENV-2. As
positive control we used Ribavirin. Compounds were considered
active against DENV-2 if has the ability to reduce or avoid the
cytopathic effect during viral infection. This assay was conducted
by quadruplicate and repeated twice for each compound.
4.4.3. Assays for pre- and post-infective stages of HHV-1, HHV-2
and DENV-2
Subsequently, the potential antiviral of active compounds was
evaluated by the plaque reduction assay as previously described
[35]. Vero cell monolayers grown in 24-well plates (2.5  104 cells/
well) were infected with 100 PFU per well of each virus. Treatments
were performed by adding compounds either simultaneously with
virus (pre-infection treatment) or after viral infection (post-infection
treatment). For pre-infection treatment, a virus/compound mixture
was added to cell monolayers and was incubated for 1 h at 37  C (5%
CO2). After incubation, washing was performed with PBS (pH ¼ 7.0)
and added CMC 1%, 0.75% and 2% for HHV-1, HHV-2 and DENV-2,
respectively. In post-infection treatment, virus was added on cell
monolayer and incubated for 1 h at 37  C (5% CO2). After, washing
was performed with PBS (pH ¼ 7.0) and compounds previously
prepared in CMC 1%, 0.75% and 2% for HHV-1, HHV-2 and DENV-2
respectively, were added. In both treatments, plates allowed to
incubate for 72 h (HHV-1 and HHV-2) and 8 days (DENV-2). Cells
were then fixed and stained with a solution of 3.5% formaldehyde
with 0.2% crystal violet and viral plaques were counted. Dextran
sulfate was employed as positive control in pre-infection assays.
Ayclovir (ACV) and Ribavirin (RIBA) were used as positive controls
in post-infection stages to HHV strains and DENV-2, respectively.
The fifty effective concentration (EC50) was defined as the con-
centration that reduces the 50% of plaque forming units. EC50 for
each compound were obtained from doseeeffect curves for linear
regression methods using the statistical GraphPad Prisma 5.0 and
EC50 values are expressed as the mean of at least four dilutions by
quadruplicate. To define which compounds were more selective to
infected cells than to non-infected cells, the antiviral selectivity
index (SI) was calculated and defined as the ratio between the
inhibitory concentration 50 (IC50) in Vero cells and the EC50 for each
virus on Vero cells.
4.4.4. Plaque size-reduction assay
For this, a PFU assay was carried out on Vero cells
(2.5  104 cells/well) and compounds previously prepared in CMC,
were added 2 h after infection with 100 PFU/well of virus [25b].
Compound 8 was added at concentrations from 1.56 mg/mL to
12.5 mg/mL and compound 9 at concentrations from 0.78 mg/mL to
6.25 mg/mL, against both HHV-1 (CDC-Atlanta and 29R) and HHV-2.
To determine the effect against DENV-2, compounds 8 and 9 were
added in a concentration range of 0.05 mg/mL to 1.56 mg/mL. Finally,
the plates were incubated for 72 h (HHV strains) and 8 days (DENV-
2), fixed and stained with a solution of 3.5% formaldehyde/0.2%
crystal violet. The images of 20 plaques for each compound con-
centration and each well were captured using a Nikon digital
camera view DS-L1 attached to a Nikon inverted microscope
(Tokyo, Japan) [25a]. The area of each plaque in millimeters was
determined using the Image-Pro Plus 6.0 software®. Two inde-
pendent experiments were performed by duplicate.
4.4.5. Cytotoxic activity
Cell growth inhibition and/or cytotoxicity on Vero, HeLa, Jurkat
and U937 cells were measured using the technique MTT. The MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
(Sigma, New Jersey, USA) assay, according to the protocol previ-
ously described [12c] with few modifications, was used. Briefly,
Vero and HeLa cells were plated at 2.0  104 cells per well in a 96-
well flat-bottomed plates and incubated for 24 h at 37  C. Moreover,
Jurkat and U937 cells were plated at 3.0  104 cells per well in a 96-
well round-bottomed plate. Then each diluted compound was
added to the appropriate wells and the plates were incubated for
further 48 h at 37  C in a humidified incubator with 5% CO2. Finally,
spectrophotometric reading was carried out at 570 nm to deter-
mine the inhibitory concentration 50 (IC50), defined as the con-
centration for each compound that inhibits 50% of cell growth. The
IC50 values for each compound were obtained by linear regression
analysis of the doseeresponse curves generated from the absor-
bance data with the statistical GraphPad Prisma 5.0. IC50 values
were expressed as the geometric mean of two independent ex-
periments done in quadruplicate. To define which compounds were
more selective to cancerous cells than to non-cancerous cells, it was
calculated selectivity index (SI) defined as Vero IC50 over HeLa,
Jurkat or U937 IC50. A compound with an IC50 < 30 mM was
considered active and with a SI  5 was considered selective.
Paclitaxel and Doxorubicine were used as positive controls.
4.4.6. Antifungal activity
The in vitro antifungal activity of compounds were evaluated
following the Clinical and Laboratory Standards Institute M38-A
protocol for filamentous fungi [36] with a few modifications and
the standard method proposed by the Antifungal Susceptibility
Testing Subcommittee of the European Committee on Antibiotic
Susceptibility Testing (AFST-EUCAST) for yeasts [37]. The filamen-
tous fungi F. oxysporum (ATCC 48112), A. fumigatus (ATCC 204305),
A. flavus (ATCC 204304), A. terreus (CDC 317) and dermatophytes, T.
rubrum (ATCC 28188) and T. mentagrophytes (ATCC 24198) were
used to evaluate antifungal activity at inoculum size of
0.2e2.5  105 CFU/mL. The yeasts C. albicans (ATCC 1023), C. par-
apsilosis (ATCC 22019) and C. tropicalis (ATCC 200956) were used to
evaluate antifungal activity at inoculum size of 1e5  105 CFU/mL.
The dilutions of compounds were dispensed into 96-well flat-
bottom microdilution plates in duplicate at final concentrations
between 50 mg/mL and 3.125 mg/mL. The compounds were
considered active when they exhibited MIC values  50 mg/mL. For
the CLSI M38-A method, the MIC were defined as the lowest dilu-
tion that resulted in an 80% of inhibition of visible growth after
incubation at 28  C to 48 h to F. oxysporum and 6 days to derma-
tophytes. Amphotericine B was evaluated, at a range of 0.031e16
mg/mL, with the strains A. fumigatus (ATCC 204305) and A. flavus
(ATCC 204304) and Terbinafine was used as positive control at a
range of 0.0078e4 mg/mL on both dermatophytes. For the AFST-
EUCAST method, the MIC was determined after 24 h of incuba-
tion at 35  C and defined as the lowest concentration that resulted
in 90% of reduction of growth. Itraconazole was used as positive
control at final concentrations of 0.031e16 mg/mL. A negative
control (inoculum without treatment) was also included. MIC
values were expressed as geometric mean (GM-MIC) of tests per-
formed in duplicate in three different assays against each fungi
species.
Competing interests
One patent application based on these results has been sub-
mitted by M.A.G. and L.A.B.-G. as inventors.
Acknowledgments
Financial support from COLCIENCIAS-Grant RC-366-2011 and
111554531592 (Patrimonio Auto
nomo del Fondo Nacional de
87
Financiamiento para la Ciencia, la Tecnología y la Innovacio
n,

de Caldas) is gratefully acknowledged. V. C. R.-L., L. S.
Francisco Jose
A.-G. and V. T.-C. thank COLCIENCIAS Programa Jo
venes Inves-
tigadores for their fellowships.
References
[1] D.D. Richman, R.J. Whitley, F.G. Hayden, Clinical Virology, third ed., ASM Press,
Washington, DC, 2009.
[2] J. Piret, G. Boivin, Antiviral drug resistance in herpesviruses other than cyto-
megalovirus, Rev. Med. Virol. 24 (2014) 186e218.
[3] J. Piret, G. Boivin, Resistance of herpes simplex viruses to nucleoside ana-
logues: mechanisms, prevalence, and management, Antimicrob. Agents Che-
mother. 55 (2011) 459e472.
[4] M.S. Mustafa, V. Rasotgi, S. Jain, V. Gupta, Discovery of fifth serotype of dengue
virus (DENV-5): a new public health dilemma in dengue control, Med. J.
Armed Forces India 71 (2015) 67e70.
[5] K.A. Hanley, S.C. Weaver, Frontiers in Dengue Virus Research, Caister Aca-
demic Press, Norfolk, 2010.
[6] TDR/WHO, Dengue: guidelines for diagnosis, treatment, Prevention and
Control, World Health Organization, Geneva, 2009.
[7] M. De Wispelaere, A.J. LaCroix, P.L. Yang, The small molecules AZD0530 and
dasatinib inhibit dengue virus RNA replication via fyn kinase, J. Virol. 87
(2013) 7367e7381.
[8] (a) J.A. Usme-Ciro, J.A. Mendez, A. Tenorio, G.J. Rey, C. Domingo, J.C. Gallego-
Gomez, Simultaneous circulation of genotypes I and III of dengue virus 3 in
Colombia, Virol. J. 5 (2008) 101;
(b) J.A. Mendez, J.A. Usme-Ciro, C. Domingo, G.J. Rey, J.A. Sanchez, A. Tenorio,
J.C. Gallego-Gomez, Phylogenetic history demonstrates two different lineages
of dengue type 1 virus in Colombia, Virol. J. 7 (2010) 226.
[9] M.A. Gonza
lez, Aromatic abietane diterpenoids: their biological activity and
synthesis, Nat. Prod. Rep. 32 (2015) 684e704.
[10] (a) M.A. Gonza
lez, Aromatic abietane diterpenoids: total syntheses and syn-
thetic studies, Tetrahedron 71 (2015) 1883e1908;
(b) M.A. Gonz
alez, Synthetic derivatives of aromatic abietane diterpenoids
and their biological activities, Eur. J. Med. Chem. 87 (2014) 834e842.
[11] G.-J. Zhang, Y.-H. Li, J.-D. Jiang, S.-S. Yu, J. Qu, S.-G. Ma, Y.-B. Liu, D.-Q. Yu, Anti-
Coxsackie virus B diterpenes from the roots of Illicium jiadifengpi, Tetrahe-
dron 69 (2013) 1017e1023.
[12] (a) L. Betancur-Galvis, C. Zuluaga, M. Arno
, M.A. Gonz
alez, R.J. Zaragoz
a,
Structure-activity relationship of in vitro antiviral and cytotoxic activity of
semisynthetic analogues of scopadulane diterpenes, J. Nat. Prod. 64 (2001)
1318e1321;
(b) M.A. Gonza
lez, Spongiane diterpenoids, Curr. Bioact. Comp. 3 (2007) 1e36;
(c) B. Zapata, M. Rojas, L. Betancur-Galvis, A.C. Mesa-Arango, D. Pe
rez-Guaita,
M.A. Gonza
lez, Cytotoxic, immunomodulatory, antimycotic, and antiviral ac-
tivities of semisynthetic 14-hydroxyabietane derivatives and triptoquinone C-
4 epimers, Med. Chem. Comm. 4 (2013) 1239e1246.
[13] J. Balzarini, E. De Clercq, B. Kaminska, A. Orzeszko, Synthesis and antiviral
activity of some 50 -N-phthaloyl-30 -azido-20 ,30 -dideoxythymidine analogues,
Antivir. Chem. Chemother. 14 (2003) 139e144.
[14] R. Bakry, R.M. Vallant, M. Najam-ul-Haq, M. Rainer, Z. Szabo, C.W. Huck,
G.K. Bonn, Medicinal applications of fullerenes, Int. J. Nanomed. 2 (2007)
639e649.
[15] E. Plummer, M.D. Buck, M. Sanchez, J.A. Greenbaum, J. Turner, R. Grewal,
B. Klose, A. Sampath, K.L. Warfield, B. Peters, U. Ramstedt, S. Shresta, Dengue
virus evolution under a host-targeted antiviral, J. Virol. 89 (2015) 5592e5601.
[16] (a) M.A. Gonz
alez, D. Pe
rez-Guaita, Short syntheses of (þ)-ferruginol from
(þ)-dehydroabietylamine, Tetrahedron 68 (2012) 9612e9615;
(b) I.M. Malkowsky, M. Nieger, O. Kataeva, S.R. Waldvogel, Synthesis and
properties of optically pure phenols derived from (þ)-dehydroabietylamine,
Synthesis 5 (2007) 773e778.
[17] Z. Zhou, Z.-X. Lin, D. Liang, J.-Q. Hu, First synthesis of ring-B C60-substituted
derivatives of N,N-(tetrachlorophthaloyl)dehydroabietylamine, Tetrahedron
69 (2013) 43e49.
[18] C.-C. Wen, Y.-H. Kuo, J.-T. Jan, P.-H. Liang, S.-Y. Wang, H.-G. Liu, C.-K. Lee, S.-
T. Chang, C.-J. Kuo, S.-S. Lee, C.-C. Hou, P.-W. Hsiao, S.-C. Chien, L.-F. Shyur, N.-
S. Yang, Specific plant terpenoids and lignoids possess potent antiviral ac-
tivities against severe acute respiratory syndrome coronavirus, J. Med. Chem.
50 (2007) 4087e4095.
[19] (a) J. Becerra, C. Flores, J. Mena, P. Aqueveque, J. Alarco
n, M. Bittner,
V. Herna
ndez, M. Hoeneisen, E. Ruiz, M. Silva, Antifungal and antibacterial
activity of diterpenes isolated from wood extractables of Chilean podo-
carpaceae, Bol. Soc. Chil. Quim. 47 (2002) 151e157;
(b) M. Fronza, R. Murillo, S. Slusarczyk, M. Adams, M. Hamburguer,
B. Heinzmann, S. Laufer, I. Merfort, In vitro cytotoxic activity of abietane
diterpenes from peltodon longipes as well as salvia miltiorrhiza and salvia
sahendica, Bioorg. Med. Chem. 19 (2011) 4876e4881.
[20] M. Bispo de Jesus, W.F. Zambuzzi, R.R. Ruela de Sousa, C. Areche, A.C. Santos
de Souza, H. Aoyama, G. Schmeda-Hirschmann, J.A. Rodriguez, A.R.M. de
Souza Brito, M.P. Peppelenbosch, J. den Hertog, E. de Paula, C.V. Ferreira,
Ferruginol suppresses survival signaling pathways in androgen-independent
human prostate cancer cells, Biochimie 90 (2008) 843e854.
88 V.C. Roa-Linares et al. / European Journal of Medicinal Chemistry 108 (2016) 79e88
[21] C. Krummenacher, A. Carfí, R.J. Eisenberg, G.H. Cohen, Entry of herpesviruses
into cells: the enigma variations, Adv. Exp. Med. Biol. 790 (2013) 178e195.
[22] D. Chattopadhyay, M.C. Sarkar, T. Chatterjee, R. Sharma Dey, P. Bag,
S. Chakraborti, M.T. Khan, Recent advancements for the evaluation of anti-
viral activities of natural products, N. Biotechnol. 25 (2009) 347e368.
[23] P. Zhong, L.M. Agosto, J.B. Munro, W. Mothes, Cell-to-cell transmission of vi-
ruses, Curr. Opin. Virol. 3 (2013) 44e50.
[24] L.T. Lin, T.Y. Chen, C.Y. Chung, R.S. Noyce, T.B. Grindley, C. McCormick, T.C. Lin,
G.H. Wang, C.C. Lin, C.D. Richardson, Hydrolyzable Tannins (Chebulagic Acid
and Punicalagin) Target Viral glycoprotein-glycosaminoglycan interactions to
inhibit herpes simplex virus 1 entry and cell-to-cell spread, J. Virol. 85 (2011)
4386e4398.
[25] (a) K. Nyberg, M. Ekblad, T. Bergstro €m, C. Freeman, C.R. Parish, V. Ferro,
E. Trybala, The low molecular weight heparan sulfate-mimetic, PI-88, inhibits
cell-to-cell spread of herpes simplex virus, Antivir. Res. 63 (2004) 15e24;
(b) M. Ekblad, B. Adamiak, T. Bergstrom, K.D. Johnstone, T. Karoli, L. Liu,
V. Ferro, E. Trybala, A highly lipophilic sulfated tetrasaccharide glycoside
related to muparfostat (PI-88) exhibits virucidal activity against herpes sim-
plex virus, Antivir. Res. 86 (2010) 196e203.
[26] K. Maringer, J. Stylianou, G. Elliott, A network of protein interactions around
the herpes simplex virus tegument protein VP22, J. Virol. 86 (2012)
12971e12982.
[27] D.C. Johnson, M. Webb, T.W. Wisner, C. Brunetti, Herpes simplex virus gE/gI
sorts nascent virions to epithelial cell junctions, promoting virus spread,
J. Virol. 75 (2001) 821e833.
[28] R.M. Mingo, J. Han, W.W. Newcomb, J.C. Brown, Replication of herpes simplex
virus: egress of progeny virus at specialized cell membrane sites, J. Virol. 86
(2012) 7084e7097.
[29] R. Dixit, V. Tiwari, D. Shukla, Herpes simplex virus type 1 induces filopodia in
differentiated P19 neural cells to facilitate viral spread, Neurosci. Lett. 440
(2008) 113e118.
[30] M.P. Taylor, O.O. Koyuncu, L.W. Enquist, Subversion of the actin cytoskeleton
during viral infection, Nat. Rev. Microbiol. 9 (2011) 427e439.
[31] J.-L. Wang, J.-L. Zhang, W. Chen, X.-F. Xu, N. Gao, D.-Y. Fan, J. An, Roles of small
GTPase Rac1 in the regulation of actin cytoskeleton during dengue virus
infection, PLoS Negl. Trop. Dis. 4 (2010) e809.
[32] M. Martínez-Gutierrez, J.E. Castellanos, J.C. Gallego-Go
mez, Statins reduce
dengue virus production via decreased virion assembly, Intervirology 54
(2011) 202e216.

, A. Lasure, D. Vanden Berghe, P.C. Rwangabo,
[33] A.J. Vlietinck, L. Van Hoof, J. Totte
J. Myukiyumwami, Screening of hundred Rwandese medicinal plants for
antimicrobial and antiviral properties, J. Ethnopharm. 46 (1995) 31e47.
[34] L.I.C. Tang, A.P.K. Ling, R.Y. Koh, S.M. Chye, K.G.L. Voon, Screening of anti-
dengue activity in methanolic extracts of medicinal plants, BMC Comple-
mentary Altern. Med. 12 (3) (2012).
[35] F.T. Cardozo, C.M. Camelini, A. Mascarello, M.J. Rossi, R.J. Nunes, C.R. Barardi,
M.M. de Mendonça, C.M. Simo ~es, Antiherpetic activity of a sulfated poly-
saccharide from agaricus brasiliensis mycelia, Antivir. Res. 92 (2011)
108e114.
[36] NCCLS, Reference method for broth dilution antifungal susceptibility testing of
conidial forming filamentous fungi, Approved standard NCCLS M38-A, Na-
tional committee for clinical Laboratory Standards, Wayne, 2002.
[37] M. Cuenca-Estrella, C.B. Moore, F. Barchiesi, J. Bille, E. Chryssanthou,
D.W. Denning, J.P. Donnelly, F. Dromer, B. Dupont, J.H. Rex, M.D. Richardson,
B. Sancak, P.E. Verweij, J.L. Rodríguez-Tudela, Multicenter evaluation of the
reproducibility of the proposed antifungal susceptibility testing method for
fermentative yeasts of the antifungal susceptibility testing subcommittee of
the European committee on antimicrobial susceptibility testing (AFST-
EUCAST), Clin. Microbiol. Infect. 9 (2003) 467e474.